Breastfeeding is associated with multiple benefits in infants;during breastfeeding, Bifidobacterium sp. are often enriched in the infant gastrointestinal tract. It has been proposed that human milk oligosaccharides (HMO), a major component of breast milk, are responsible for this enrichment. There are more than 200 HMOs, while only 40 bovine milk oligosaccharides (BMO) are observed in bovine milk;however, it was shown that several HMO constituents are conserved with BMO. Previous work has demonstrated that HMO are uniquely utilized by probiotic bacteria Bifidobacterium longum subsp. infantis ATCC 15697 (B. infantis). Other bifidobacteria found in fecal samples of breast-fed infants, e.g. B. longum subsp. longum, B. breve and B. bifidum utilize different mechanisms of HMO strategies and show significantly slower growth on HMO as only carbon source. Beneficial bacteria (probiotics) have both direct and indirect effects on the host, including enhancement of epithelial barrier function, modulation of the mucosal immune system, and alteration of the intestinal microbiota. The binding of bacteria to the mucus layer is a prerequisite for adhesion and prevents the bacteria from being removed by intestinal peristalsis. Following adhesion, probiotics start to interact with the environment. In newborns the integrity of the epithelial layer is not complete and an increased permeability to bacteria and luminal antigens can potentially trigger mucosal inflammation. We predict that unique HMO structure allows a very specific relationship between bifidobacteria (especially B. infantis) and intestinal epithelial cells compared to other bifidobacteria. This application is designed to test the hypothesis that growth on HMO stimulates the interaction between B. infantis and infant's gut including adhesion, prevents permeability and translocation and affects the expression of tight junction proteins. Furthermore, we hypothesize that HMO-grown B. infantis induces an anti- inflammatory immune response in the epithelial cells and plays a role in maturation of the immune system compared to other bifidobacteria. These studies will generate knowledge of the impact of milk oligosaccharides on microbial function in intestine. Also, the results could advance the understanding of how infant formulas could be supplemented with specific oligosaccharides to increase positive action of bifidobacteria in the infant's developing gut. This application concentrates on efforts likely to yield a great impact on the health and well-being of infants. Successful completion of these experiments will provide a detailed functional understanding of the mechanism of action of beneficial endogenous bacteria together with the specific substrates that promote their competitive growth and regulate their function. PUBLIC HEALTH RELEVANCE: This application concentrates on efforts likely to yield a great impact on the health and well-being of infants. These novel studies will fill the knowledge gap regarding the impact of human milk oligosaccharides on cognate microbial function in intestine and serve as a model of how diet modulates the interface between microbes and intestinal epithelium. Successful completion of these experiments will provide a detailed functional understanding of the mechanisms of action of beneficial endogenous bacteria together with the specific food substrates that promote their competitive growth and regulate their function.